Identification of key residues for activities of atypical glutathione S-transferase of Ceriporiopsis subvermispora, a selective degrader of lignin in woody biomass, by crystallography and functional mutagenesis.

Ceriporiopsis subvermispora (C. subvermispora) is a selective degrader of lignin in the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in cellular detoxification and metabolism. The crystal structures of a GST of C. subvermispora, CsGST83044, in GSH-free and -bound forms were solved at 1.95 and 2.19 Šresolution, respectively. The structure of the GSH-bound form revealed that CsGST83044 can be categorized as an atypical-type of GST. In the GSH-bound form of CsGST83044, Asn22, Asn24, and Tyr46 are located closest to the sulfur atom and form hydrogen bonds with the thiol group. The functional mutagenesis indicated that they are critical for the enzymatic activities of CsGST83044. The critical residues of an atypical-type GST belonging to the GSTFuA class were revealed for the first time. A previous study indicated that CsGST83044 and another GST, CsGST63524, differ in substrate preference; CsGST83044 prefers smaller substrates than CsGST63524 for its esterase activity. The GSH-bound pocket of CsGST83044 turns out to be small, which may explain the preference for smaller substrates. Protein engineering of GSTs of C. subvermispora in the light of the obtained insight may pave a path in the future for utilization of the woody biomass.

Characterization of the glutathione S-transferases that belong to the GSTFuA class in Ceriporiopsis subvermispora: Implications in intracellular detoxification and metabolism of wood-derived compounds.

Glutathione S-transferases (GSTs) of wood-degrading fungi play essential roles in cellular detoxification processes and endogenous metabolism. Fungal GSTs of GSTFuA class are suggested to be involved in lignin degradation. Ceriporiopsis subvermispora is one of the important model fungi of the selective lignin degraders, we found it interesting to study its GSTs. Here, we characterized the activities of two GSTs of the GSTFuA class of C. subvermispora (CsGST63524 and CsGST83044). A high-yield expression systems involving Escherichia coli was developed for each of these enzymes. Both enzymes were found to exhibit GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene, and GSH-peroxidase activity toward cumene hydroperoxide. Both enzymes showed high GSH-conjugation activity under basic conditions (pH8.0 to 9.0), and the optimum temperature for their activity was 40°C. In addition, three fluorescent compounds were used i.e., methylumbelliferyl acetovanillone was used to monitor etherase activity, and 5-chloromethylfluorescein diacetate and 4-methylumbelliferyl acetate to monitor esterase activity. CsGST83044 exhibited both etherase and esterase activities, while CsGST63524 displayed only esterase activity, which was much higher than that of CsGST83044. These findings imply the functional diversity of the GSTFuA class GSTs of C. subvermispora, suggesting that each protein plays distinctive roles in both the fungal detoxification system and wood compound metabolism.

Simultaneous removal of AOX and COD from real recycled paper wastewater using GAC-SBBR.

A lab-scale granular activated carbon sequencing batch biofilm reactor (GAC-SBBR), a combined adsorption and biological process, was developed to treat real wastewater from a recycled paper mill. In this study, one-consortia of mixed culture (4000-5000 mg/L) originating from recycled paper mill activated sludge from Kajang, Malaysia was acclimatized. The GAC-SBBR was fed with real wastewater taken from the same recycled paper mill, which had a high concentration of chemical oxygen demand (COD) and adsorbable organic halides (AOX). The operational duration of the GAC-SBBR was adjusted from 48 h to 24, 12 and finally 8 h to evaluate the effect of the hydraulic retention time (HRT) on the simultaneous removal of COD and AOX. The COD and AOX removals were in the range of 53-92% and 26-99%, respectively. From this study, it was observed that the longest HRT (48 h) yielded a high removal of COD and AOX, at 92% and 99%, respectively.